This invention relates to a new group of specific, irreversible inhibitors of acetyl-cholinesterase (International Enzyme classification EC3.1.1.7) and to methods of making and using these inhibitors.
Acetylcholinesterase is a key enzyme that catalyzes the hydrolysis of acetylcholine to choline and acetic acid via an acyl enzyme intermediate. Along with choline acetyl transferase, acetylcholinesterase regulates the synthesis and metabolism of acetylcholine. Acetylcholinesterase is essential for ganglionic and interneuronal transmission in several parts of the central nervous system.
A number of common abnormalities and physical conditions are characterized by a dysfunction of the production and/or metabolism mechanisms of acetylcholine. Myasthenia gravis, neurological disorders, horse colic, ophthalmological disorders, miotic induction and drug induced extra-pyramidal symptoms which accompany psychiatric drug therapy respond to therapeutic intervention of the cholinergic system. Acetylcholinesterase inhibitors also find uses in the agricultural market as pesticides.
The dysfunction in the production or metabolism mechanisms of acetylcholinesterase can result in excess or deficient levels of this key enzyme substrate in the organism. Inhibitors of acetylcholinesterase may be useful in the treatment and diagnosis of acetylcholinesterase abnormalities, limited by the side effects associated with the administration of the inhibitor under study.
A number of irreversible enzyme inhibitors are known. However, few of them have gained clinical significance, in large part, because of their low specificity for a particular enzyme and their high toxicity.
Known irreversible acetylcholinesterase inhibitors include organophosphorous compounds (see, W. N. Aldridge & E. Reiner, Enzyme Inhibitors as Substrates. Interaction of Esterases with Esters of Organophosphorous and Carbamic Acids pp. 1-328 (North Holland, Amsterdam)(1972)), organocarbamates, (see, Aldridge & Reiner, supra), sulfonates (see, D. K. Myers & A. Kemp, Nature, 173, pp. 33-34 (1954)), and arsonates (see, R. Kitz & I. B. Wilson, J. Biol. Chem. 237, pp. 3245-49 (1962)). While these irreversible inhibitors bind to the active site of acetylcholinesterase, they also tend to phosphorylate, carbamylate, sulfonate or arsonate any other proteases or esterases which can form an acyl enzyme complex. It is this inactivation of these other enzymes which produces many of the undesirable side effects associated with the use of these inhibitors. In addition, this non-specific binding on other proteases and esterases consumes the inhibitor, thus necessitating larger and more frequent doses of the inhibitor, accompanied again by the undesirable side effects of such treatment.
Other inhibitors of acetylcholinesterase include Methacholine Chloride, Methacholine Bromide, Carbachol, and Bethanechol Chloride (see, Textbook of Organic Medicinal and Pharmaceutical Chemistry, (C. Wilson, O. Griswold & R. Doerge eds.) (J. B. Lippincott Co. Philadelphia) pp. 507-10 (1971)). These inhibitors have gained some clinical significance, however the side effects associated with their administration still present clinical problems.
Acetylcholinesterase can be inhibited irreversibly by a group of phosphate esters that are highly toxic. While these inhibitors have marginal clinical usage, until now limited to human clinical experiments for myasthenis gravia, they have been used extensively as insecticides. Two examples of such esters include hexaethyltetraphosphate and tetraethyl pyrophosphate. (see, Textbook of Organic Medicinal and Pharmaceutical Chemistry pp. 507-10, supra). A problem with these existing acetylcholinesterase inhibitors that are used as insecticides is their general insolubility in petroleum ethers and kerosene. Although somewhat soluble in water, they are quickly deactivated by hydrolysis. This insolubility in the normal spraying oils and instability in water makes application of these insecticides more difficult. (Id. at 510).
Halomethylated derivatives of dihydrocoumarins react biologically on alpha-chymotrypsin in much the same way that the alpha substituted cresol choline carbonates, provided by this invention, react biologically with acetylcholinesterase. The two inhibitors, however, show few structural similarities. (see, Bechet, DuPaix & Blagoeva, Inactivation of alpha-chymotrypsin by new bifunctional reagents: halomethylated derivatives of dihydrocoumarins, Biochemie, 59, 231 (1977)).
Notwithstanding the foregoing, the art has not heretofore taught or suggested an acetylcholinesterase inhibitor that was more specific and of lower systemic toxicity than existing inhibitors; nor has the art suggested an inhibitor of the structural class of this invention.